Regulation of the carbon flux in Synechocystis using the PGAM-PirC Switch

Cyanobacteria are autotrophic organisms, which perform oxidative photosynthesis. Their ability to oxidize water and use the electrons for reducing carbon dioxide (CO2) to form organic matter by using solar energy makes them an interesting organism for biotechnological applications. Therefore, Synechocystis sp. PCC 6803 is studied as a model organism for the construction of green cell factories.

An important control point of the carbon flux is the 2,3-bisphosphoglycerate-independent phosphoglycerate-mutase (PGAM), which converts the first CO2 fixation product 3-phosphoglycerate to 2-phosphoglycerate. This reaction directs carbon flow towards lower glycolysis for the production of amino acids, fatty acids, biopolymers, such as polyhydroxybutyrate (PHB) and more. PGAM activity is inhibited through binding of the small protein PirC, which itself is repressed by the PII protein, the sensor protein for the carbon/nitrogen status [1].

The aim is to use this PGAM-PirC key hub to direct the metabolic flux towards lower glycolysis and the production of PHB and other feedstock chemicals. Therefore, a strain constitutively overexpressing pgam was constructed and the level of PGAM and glycogen, PHB production and metabolite excretion were analyzed. In addition, several deletion mutants were tested, e.g., a ΔphaEC mutant, which cannot produce PHB. Furthermore, we analyzed the PGAM enzyme biochemically and identified structural elements required for regulation by PirC [2]. Strains with the corresponding PGAM variants were also constructed.

Our results revealed that PGAM overproduction led to lower glycogen levels, whereas PHB amounts were higher under nitrogen depletion compared to the WT. Deletion of pirC or phaEC resulted in higher concentrations of the tested metabolites pyruvate, succinate and 2-oxoglutarate extracellularly. The double mutant lacking pirC and phaEC showed the highest excretion of the tested metabolites.

Our work shows the important role the PGAM-PirC switch is playing in the metabolism. By tuning this regulation further, a platform will be established to redirect carbon flow for enhanced valuable chemical production.

[1] Orthwein et al., 2021 Proc. Natl. Acad. Sci. U. S. A., 10.1073/pnas.2019988118

[2] Orthwein et al. 2024 www.biorxiv.org/content/10.1101/2024.10.28.619893v1